The objective of this research is to explore the biochemical events that lead from nuclear quiescence to mitogenesis using epidermal growth factor (EGF) as the activating agent. We have characterized the EGF-receptor from both mouse and human as a cell-surface polypeptide of Mr 170,000-180,000. We have antireceptor antibodies directed against the human receptor. Also we have demonstrated spontaneous and selective transfer of exogenous EGF-receptors from donor membranes to receptor-negative variant cells. These techniques and others (receptor fragmentation, photochemical crosslinking) will be exploited to investigate the receptor molecule domains that are involved in crucial interactions with other proteins in the membrane at the beginning of the mitogenic pathway. We will also use an insulin-receptor system as a model to study receptor development and expression in vitro. Regarding the intracellular events that are induced by EGF, we have described an EGF-induced cytoplasmic protein that can stimulate DNA synthesis in a cell-free nuclear assay. This in vitro assay will be used to study the protein factors that regulate entry into the DNA synthetic phase. A method for measuring loss of commitment to DNA replication has been developed by us, and our studies suggest that commitment is a whole-cell state that can decay in a single step. We plan to use 2-dimensional gel electrophoresis to identify proteins whose appearance or decay can be correlated with the cell's commitment or loss of commitment to DNA synthesis. In summary, our goal is to study molecular events at the receptor end of the mitogenic pathway, among intermediates, and at the final commitment stage. Specific aims are directed at each of these stages.